以顳葉型癲癇動物模式探討orexin/hypocretin於睡眠日夜節律所扮演的角色

The Role of Orexin/hypocretin on Circadian Rhythm in a Temporal Lobe Epilepsy (TLE) Rat

陳盈如(Ying-Ju Chen)

指導教授 ： 張芳嘉

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01686

摘要

癲癇是一種復發性痙攣的慢性異常病症，原因為腦部神經細胞，突然且短暫地過度放電所導致。臨床與實驗結果顯示，睡眠與癲癇的關係相當複雜且交互影響。事實上，癲癇患者常有夜間失眠、睡眠片段化及白天嗜睡之症狀，而導致如此的機制可能與睡眠恆定及日夜節律有關係。我們於大鼠之杏仁核給予電刺激，誘導顳葉癲癇，模擬顳葉癲癇患者睡眠-清醒週期改變的情形。管理生物體生理及行為之生物時鐘座落於下視丘之上視核 (suprachiasmatic nucleus；SCN)。構成哺乳類節律時鐘的成因為SCN細胞內節律基因之負回饋調節，其中，per1基因於subjective day表現最高，而bmal1相對於subjective night表現較高。這些節律基因在睡眠-清醒週期的節律上扮演著重要的角色。Orexin/hypocretin為一新興之激素，中樞神經系統內分泌orexin/hypocretin的神經細胞主要位於側下視丘，該部位亦受到杏仁核中心核之投射。近期有報告證實SCN上有orexin/hypocretin receptors，並在體外實驗發現orexin/hypocretin可改變SCN之放電活性。故我們將藉由阻斷orexin receptor 1 / hypocretin receptor 1，了解在TLE實驗模式下，orexin/hypocretin是否扮演了癲癇與睡眠-清醒週期改變的中介因子。並比較正常大鼠與顳葉癲癇大鼠SCN內節律蛋白PER1的表現。
適應12:12小時亮暗週期之Sprague-Dawley公鼠在亮期中點circadian time 6 (CT 6)接受杏仁核電刺激，誘導full-blown epilepsy；電刺激前於SCN內微量注射orexin receptor 1 / hypocretin receptor 1拮抗劑—SB334867。以腦電波記錄及影像記錄分析其睡眠-清醒變化。另外，以免疫組織化學染色呈現正常大鼠與顳葉癲癇大鼠在CT 6接受電刺激後之SCN內PER1節律蛋白的變化。
顳葉癲癇大鼠與先前對照處置相比較：於CT 23 – 24，非動眼睡眠量由9.56 ± 3.37 %顯著上升至27.36 ± 4.71 %，清醒量則有相對顯著下降；而於接下來的亮期最末兩小時內CT 11 - 12，非動眼睡眠量由42.81 ± 4.16 %顯著下降到18.97 ± 3.15 %，清醒量亦有相對顯著上升。而這些變化，在電刺激之前於SCN微量注射SB334867，會被反轉回到正常數值。SB334867單獨施打於SCN則對睡眠-清醒週期未產生影響。在節律蛋白PER1的表現量，顳葉癲癇大鼠與對照組SCN內PER1蛋白質之表現高峰分別出現在CT 6以及CT 12。於CT 6時間點，兩組之表現量分別為358 ± 64個與147 ± 14個，有顯著差異。
結果顯示，於CT 6以杏仁核電刺激誘導full-blown seizures，會造成日夜節律的改變，而有兩小時之前移。而此一效應藉由於SCN內注射orexin receptor 1 / hypocretin receptor 1拮抗劑被阻斷，因此此日夜節律之改變，orexin/hypocretin的確涉及其中。

關鍵字

顳葉癲癇 ； 睡眠 ； 日夜節律 ； 節律蛋白PER1 ； orexin/hypocretin

並列摘要

Epilepsy is a chronic disorder characterized by recurrent seizures, which may vary from a brief lapse of attention or muscle jerks to severe and prolonged convulsions. It was caused by sudden, brief, excessive electrical discharges in a group of brain cells. Clinical and experimental observations suggest that the relationship between sleep and epilepsy is reciprocal. Excessive daytime sleepiness and sleep complaints are common among epilepsy patients, which implies the influence of epilepsy on circadian rhythm. However, little is known about this interaction. Amygdala kindling-induced temporal lobe epilepsy (TLE) in rats was used in current study to mimic the patients with epilepsy. Suprachiasmatic nucleus (SCN) of anterior hypothalamus is the pacemaker controlling circadian rhythm. The circadian oscillation is driven by clock genes in the SCN, including per1, per2, per3, cry1, cry2, CLOCK, and bmal1. Circadian oscillation of per1 peaks at subjective day and in antiphase to bmal1 expression, which peaks at subjective night. In addition, the lateral hypothalamic area (LHA), which received afferents from the central nucleus of amygdala (CeA), involves in the homeostatic regulation of sleep-wake rhythmicity. The neuropeptide secreted form LHA is orexin/hypocretin, which is as a waking promoter. Moreover, orexinergic/hypocretinergic fibers and receptor expressions are detected in the SCN region. Therefore, we investigated the role of hypocretin in TLE-induced sleep alternations by pharmacological blockade of hypocretin receptor 1 and determined the expressions of PER1 in SCN between the normal and TLE rats.
Male Sprague-Dawley rats were housed in a 12:12-hour light:dark cycle. Kindling stimuli delivered via a bipolar electrode, placing in the right central nucleus of the amygdala, at circadian time 6 (CT 6). We injected orexin receptor 1 / hypocretin receptor 1 antagonist, SB334867, into the SCN of the TLE rats. Electroencephalogram (EEG) and movement-defined sleep-wake activity were collected. The other group, rats received a single stimulus at CT6. At the following circadian time points, CT18 and the next CT0, CT3, CT6, CT9 and CT12, normal and TLE rats were sacrificed and their brains were processed for immunohistochemical detection of PER1.
The amount of non-rapid eye movement sleep (NREMS) after CT 6 amygdala-kindling was increased from 9.56 ± 3.37 % after vehicle to 27.36 ± 4.71 % during the CT23-24. There was a mirrored reduction in the amount of wakefulness. The amount of NREMS during the subsequent CT11-12 was decreased from 42.81 ± 4.16 % after vehicle to 18.97 ± 3.15 % after CT6 amygdala-kindling. There was also a mirrored increase in the amount of wakefulness. The alterations of NREMS after CT 6 amygdala-kindling were blocked when orexin receptor 1 / hypocretin receptor 1 antagonist, SB334867, was administered. Besides, SB334867 administration at CT 6 in normal rats didn’t have effect on spontaneous NREMS. In addition, the control group exhibited maximal values for PER1 at CT 12, and the CT 6 kindling group peaked at CT 6. PER1-positive cells in the CT 6 kindling group increased from 358 ± 64 to 147 ± 14 compared to the control group at CT 6.
The CT 6 amygdala kindling results in the circadian phase shifts with a 2-hour advance. However, the circadian alteration was blocked by microinjection of hypocretin receptor1 antagonist into SCN. This result suggests that circadian phase shift induced by CT 6 amygdala kindling is mediated by orexin/hypocretin.

並列關鍵字

temporal lobe epilepsy (TLE) ； sleep ； circadian rhythm ； PER1 ； orexin/hypocretin

參考文獻

1. Proposal for revised clinical and electroencephalographic classification of epileptic seizures. From the Commission on Classification and Terminology of the International League Against Epilepsy. Epilepsia 22: 489-501, 1981.

2. Agostino PV, Ferreyra GA, Murad AD, Watanabe Y, and Golombek DA. Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei. Neurochem Int 44: 617-625, 2004.

3. Ahmed I and Thorpy M. Clinical features, diagnosis and treatment of narcolepsy. Clin Chest Med 31: 371-381.

4. Aldrich MS. Diagnostic aspects of narcolepsy. Neurology 50: S2-7, 1998.

5. Antoch MP, Song EJ, Chang AM, Vitaterna MH, Zhao Y, Wilsbacher LD, Sangoram AM, King DP, Pinto LH, and Takahashi JS. Functional identification of the mouse circadian Clock gene by transgenic BAC rescue. Cell 89: 655-667, 1997.